1. A Study On Spatial Heterogeneity Of Infiltrating T Lymphocytes In Primary Liver Cancer 2. A Study On Relationship Between The Detection Of Circulating Tumor Cells And Outcomes In Patients With Primary Liver Cancer

Part I A study on spatial heterogeneity of infiltrating T lympho-cytes in primary liver cancerBackground:Primary liver cancer(PLC)also known as liver cancer which originates from liver cells or intrahepatic bile duct epithelial cells is one of common malignant tumor in our country.Liver cancer is the third and second leading cause of cancer-related death among both men and women in China and worldwide,respectively.Despite substantial improvements in screening,diagnosis and treatment,the prognosis of PLC patients remains poor.Immunotherapeutic approaches based on tumor infiltrating lymphocytes(TILs)have demonstrated that durable responses are produced in some patients with solid tumors.Besides,preliminary evidence suggests that Nivolumab,a programmed cell death protein-1(PD-1)immune checkpoint inhibitor,had a manageable safety profile and durable objective responses on patients with advanced hepatocellular carcinoma,which suggests the potential feasibility of clinical application of immunotherapy for PLC.However,the spatial distribution of TILs,local gene signature and mutation burden remain unexplored in PLC.Therefore,it is necessary to dissect the interaction between TILs and tumor cells.Aim:In order to assess the spatial distribution of TILs,and the relationship between TIL diversity,local immune response and mutation burden in PLC,we performed immune repertoire sequencing,gene expression profiling analysis and whole-exome sequencing in parallel on five regions of each tumor and on matched adjacent normal tissues and peripheral blood from five PLC patients.Methods and materials:Our study included tumor,adjacent normal tissue and peripheral blood specimens collected from five patients diagnosed with PLC at the time of hepatectomy,every tumor tissue included five sites.The proportion of tumor cells was evaluated by HE staining.RNA was extracted directly and TCRP complementary detennining region 3(CDR3)region was amplified by Multiplex PCR and sequenced by Illumina Hiseq 2500 platform.Besides,RNA of tumor samples were examined with Agilent whole genome gene expression microarray.And DNA of tumor samples were used to exon trapping and then sequenced on an Illumina HiSeq 2000 instrument.To evaluate the contribution of sampling noise and sequencing error introduced by PCR and sequencing method,we performed replicate sequencing reactions in all five tumor samples from Patient 4.Results:The total number of unique TCR? reads achieved for each sample type from the five patients was 2.55×105 to 9.88×105for tumor tissues,3.33×105 to 7.51×105 for adjacent noncancerous liver tissues,and 4.74×105 to 1.27×106 for peripheral blood,which indicated that peripheral blood contains more T cell clone types than tumors or adjacent noncancerous liver tissues(blood vs tumor,p<0.001;blood vs noncancerous tissues,p = 0.007).A significantly higher cumulative frequency of the top 250 most abundant TIL clones was observed in tumors than in peripheral blood(p =0.039).Besides,overlap rates of T cell receptor(TCR)repertoire for intratumor comparisons,significant higher than those for tumor-adjacent normal tissue comparisons and tumor-blood comparisons,which provide evidence for antigen-driven clonal expansion in PLC(tumor-tumor pairs vs tumor-adjacent normal tissiue pairs,p<0.001;tumor-tumor pairs vs tumor-peripheral blood pairs,p<0.001,).Only 7.92%-24.07%(median:10.93%)of the 250 T cell clones detected with the highest frequency in each tumor sample were ubiquitous,whereas 75.93-92.08%(median:89.07%)of the T cell clones were heterogeneous.Correlation analysis revealed that TIL diversity significantly correlated with T cell infiltration score(TIS)and the overall immune infiltration score(IIS)(ShannonDI vs TIS=0.852,p<0.001;ShannonDI vs TIS=0.798,p<0.001).Besides,we found the PD-1 and the antigen presenting machinery(APM)were higher in tumor sites which acquired higher ShannonD1 and TIS(ShannonDI vs PD-1=0.613,p=0.001;TIS vs PD-1 =0.641,p=0.001;ShannonDI vs APM=0.574,p=0.003;TIS vs PD-1=0.56,p=0.004).While,TIL diversity and APM had no connection with the mutational load.Conclusions:(1)T cell clones within tumor microenvironment in PLC were tumor-relate(2)TIL populations found in PLC were spatially heterogeneous.(3)TIL diversity and PD-1 identified in PLC were spatially heterogeneous,indicated tumor microenvironment plays an important role in therapeutic regimen making.(4)Improve APM in tumor tissues is the key to success in immunotherapy.Part II A study on relationship between the detection of circulating tumor cells and outcomes in patients with primary liver cancerBackground:Primary liver cancer is one of the most common cancers in worldwide.Currently,the clinical treatment of liver cancer are mainly hepatectomy,liver transplantation,interventional therapy,radio frequency ablation,radiation and chemo-therapy,molecular targeting and immunotherapy,the preferred treatment for the hepatectomy and liver transplant.The research found that the final bottleneck of improving the postoperative survival in patients with PLC are inevitable postoperative recurrence and metastasis.Therefore,recurrence and metastasis of liver cancer have become an important factor affecting the prognosis of patients with liver cancer.The present clinical study primarily by imageological examination and serum markers for tumor diagnosis,treatment and monitoring,but the results are not satisfactory.Since the concept of circulating tumor cell(CTCs)was proposed,CTCs gradually entered into the view of more scholars.Since CTCs are part of the primary tumor,and they have similar and even the same characteristic,some scholars believe it may be CTCs as a representative of the original tumor "liquid biopsy specimens" or ""marker".With a systematic and in-depth study of CTCs in the process of recurrence and metastasis of tumor,we found that if the effective way for tumor patients for detection of CTCs in peripheral blood,it can not only help the early diagnosis and treatment monitoring,but also can effectively predict the prognosis.Aim:Analyze the dynamic change of CTCs in course of disease and the relationship between CTCs and TNM staging,AFP level and recurrence(within 6 month),and so on,to further explore the CTCs detected in the clinical diagnosis and treatment of primary liver cancer application.Methods and materials:We used imaging flowcytometry to verify whether oHSVI-hTERT-GFP which established by our laboratory research group could specifically capture the CTCs in peripheral blood.Then,selected in September 2015 to October 2016 the National Cancer Center/Hospital of the Chinese Academy of Medical Sciences and Peking Union Medical College hepatobiliary surgery treated 60 cases of primary liver cancer were enrolled.The peripheral blood of patients was collected at preoperative,the 7th day after operation and 3 months after the operation,respectively.Then CTCs were detected with oHSVI-hTERT-GFP,analyze it with AFP level,cirrhosis,hepatitis B surface antigen,number of tumors,tumor size,TMN stage,degree of differentiation,with or without vascular thrombosis,recurrence,with or without clinical pathology relations.Besides,analyze the dynamic change of CTCs in course of disease,to further explore the CTCs detected in the clinical diagnosis and treatment of primary liver cancer application.Results:Imaging flowcytometry successfully identified the cells with CD45-,GFP+,and GPC3+ in peripheral blood.60 patients which eligibility was included in this study,all of them detected the CTCs before operation,42 patients and 35 patients detected the CTCs at the 7th day after operation and 3 months after the operation,respectively.And the positive rate were 100%?100%and 91.43%(32/35),respectively.Of 60 patients,34 patients detected CTCs at all three time point.The number of preoperative CTCs in patients with the number of tumors and microvascular thrombosis was statistically associated,but the CTCs detected at the 7th day and 3 months after the operation.The number of CTCs detected at the 7th day following surgery was significantly higher than that before surgery,and the number of CTCs at 3 months after operation was significantly lower than that of the 7th day after operation,but there was no significant difference before operation.The dynamic change of CTCs in course of disease was statistically according to the TNM staging,the number of peripheral blood CTCs in the stage ?+? is gradually increasing as the disease progresses.Conclusions:(1)The number of preoperative CTCs in patients with the number of tumors and microvascular thrombosis was statistically associated.(2)The number of peripheral blood CTCs in the stage ?+? is gradually increasing as the disease progresses.